FEBS Letters 587 (2013) 2018–2024

journal homepage: www.FEBSLetters.org

Review In search of novel highly active mitochondria-targeted : Thymoquinone and its cationic derivatives

Inna I. Severina a,1, Fedor F. Severin a, Galina A. Korshunova a, Natalya V. Sumbatyan a, Tatyana M. Ilyasova a, Ruben A. Simonyan a, Anton G. Rogov b, Tatyana A. Trendeleva b, Renata A. Zvyagilskaya b, Vera B. Dugina a, Lidia V. Domnina a, Elena K. Fetisova a, Konstantin G. Lyamzaev a, Mikhail Yu Vyssokikh a, Boris V. Chernyak a, Maxim V. Skulachev a,e, ⇑ Vladimir P. Skulachev a,c, , Viktor A. Sadovnichii d a Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, Institute of Mitoengineering, Vorobyevy Gory 1, Moscow 119992, b Bach Institute of Biochemistry, Russian Academy of Science, Leninsky prosp. 33, Moscow 119071, Russia c Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, Russia d Lomonosov Moscow State University, Faculty of Mechanics and Mathematics, Russia e Lomonosov Moscow State University, Faculty of Biology, Department of Virology, Russia article info abstract

Article history: Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many Received 23 April 2013 human pathologies. Thymoquinone (2-demethylplastoquinone derivative) was identified as an Accepted 30 April 2013 active component of this extract. Recently, it was shown that conjugates of plastoqui- Available online 10 May 2013 none and penetrating cations are potent mitochondria-targeted antioxidants effective in treating a large number of age-related pathologies. This review summarizes new data on the antioxidant Edited by Alexander Gabibov, Felix Wieland and Wilhelm Just and some other properties of membrane-penetrating cationic compounds where 2-demethylplasto- quinone substitutes for . It was found that such a substitution significantly increases a window between anti- and prooxidant concentrations of the conjugates. Like the original plastoqui- Keywords: Thymoquinone none derivatives, the novel compounds are easily reduced by the respiratory chain, penetrate SkQ through model and natural membranes, specifically accumulate in mitochondria in an electropho- Mitochondria-targeted antioxidant retic fashion, and strongly inhibit H2O2-induced apoptosis at pico- and nanomolar concentrations in Apoptosis cell cultures. At present, cationic demethylplastoquinone derivatives appear to be the most promis- Reactive oxygen species ing mitochondria-targeted drugs of the quinone series. Cancer Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

1. Quinones as mitochondria-targeted antioxidants

Mitochondria are the only intracellular organelles whose inte- rior is negatively charged relatively to the exterior [1,2]. This fact Abbreviations: Dw, transmembrane electric potential difference; BLM, bilayer can be used to specifically address various compounds to mito- planar membrane; C12R1, dodecyl rhodamine 19; C12TPP, dodecyltri- phenylphosphonium; DMMQ, 30-demethoxyMitoQ; MDA, malondialdehyde; MDR, chondria. To this end, it was suggested to combine the transported multidrug resistance; MitoQ, 10-(60-ubiquinonyl)decyltriphenylphosphonium; compound with a positively charged ion easily penetrating ROS, reactive oxygen species; SkQ1, 10-(60-plastoquinonyl)decyltriphenylphospho- through biomembranes [3,4]. To make an ion permeable for mem- nium; SkQ3, (60-methylplastoquinonyl) decyltriphenylphosphonium; SkQR1, branes, its ionized atom should be surrounded by bulky hydropho- 10-(60-plastoquinonyl) decylrhodamine 19; SkQT1(p), 10-(60-toluquinonyl) decyl- triphenylphosphonium; SkQT1(m), 10-(50-toluquinonyl) decyltriphenylphosphoni- bic residues that delocalize the electric charge of this atom [1,2,5]. um; SkQT1, a mixture of SkQT1(p) and SkQT1(m) in proportion of 1.4:1; SkQTR1, Such a principle was employed to construct mitochondria-targeted 10-(60-toluquinonyl) decylrhodamine 19; TQ, thymoquinone antioxidants [5–18]. Among them, some quinone derivatives ⇑ Corresponding author at: Lomonosov Moscow State University, Belozersky proved to be the most active (Fig. 1). As was found in our group, Institute of Physico-Chemical Biology, Vorobyevy Gory 1, Moscow 119992, Russia. Fax: +7 4959393181. the antioxidant activity measured in isolated mitochondria treated 2+ 0 E-mail address: [email protected] (V.P. Skulachev). with Fe and ascorbate increases in the series: 10-(6 -ubiquino- 1 Died on Nov. 9, 2012. F.F.S., G.A.K., N.V.S., T.M.I., R.A.S., A.G.R., T.A.T., R.A.Z., V.B.D., nyl)decyltriphenylphosphonium (MitoQ) < 30-demethoxyMitoQ L.V.D., E.K.F., K.G.L., M.Y.V., B.V.C., M.V.S., V.P.S. and V.A.S. dedicate this paper to our (DMMQ) = (60-methylplastoquinonyl) decyltriphenylphosphonium unforgettable Inna whose last experimental results are included in the article.

0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.04.043 I.I. Severina et al. / FEBS Letters 587 (2013) 2018–2024 2019

(SkQ3) < 10-(60-plastoquinonyl)decyltriphenylphosphonium 2. Cationic thymoquinone derivatives: effect on model (SkQ1) [15,16]. Thus, substitutions of methoxy group by methyl membranes, isolated mitochondria and cell cultures and by an H atom seem to be favorable for antioxi- dant activity. It would be interesting to continue this quinone ser- SkQT1 and SkQTR1 were synthesized in essentially the same ies by substituting one more methyl group in plastoquinone by an way as their plastoquinone analogs, SkQ1 and 10-(60-plastoquino- H atom, just as it occurs in 2-demethylplastoquinone, an interme- nyl) decylrhodamine 19 (SkQR1) [15]. SkQT1 samples were a mix- diate of plastoquinone biosynthesis [19] and in so-called thymoqui- ture of p and m isomers (Fig. 1B) in the proportion of 1.4:1. SkQTR1 none (Fig. 1A), a plant antioxidant responsible for many favorable was purified as p isomer, like other SkQs [15]. pharmacological effects of black cumin (see below, Section 3). In the first series of experiments, generation of diffusion poten- We studied thymoquinone-like derivatives conjugated with pene- tial of SkQT1 on bilayer planar phospholipid membranes (BLM) trating cations, namely decyltriphenylphosphonium (in SkQT1) was demonstrated. Like other penetrating cations [2,4], the and decylrhodamine 19 (in 10-(60-toluquinonyl) decylrhodamine concentration gradient of SkQT1 was found to generate an 19 (SkQTR1)) (Fig. 1B). In the next section, some results of this electric potential difference, the compartment with lower study are reviewed. [SkQT1] being positively charged due to downhill transmembrane

Fig. 1. Formulas of certain compounds considered in this review. (A) Mitochondria-targeted cationic quinone derivatives and thymoquinone. R, decyltriphenylphosphonium. (B) Cationic quinol derivatives of SkQ series and their analogs lacking quinol residue. 2020 I.I. Severina et al. / FEBS Letters 587 (2013) 2018–2024 diffusion of this cation. The magnitude of transmembrane electric High (micromolar) levels of both SkQ1 and SkQT1 decrease potential difference (Dw) reached the theoretical value (about membrane potential in animal (Fig. 3E) and (not shown) 60 mV per 10-fold concentration gradient of the penetrating - mitochondria. Moreover, both compounds strongly potentiated a ion, Fig. 2A). Dw decrease caused by palmitate (Fig. 3F), an effect accounted When SkQTR1 was used instead of SkQT1, the BLM responses for by facilitating palmitate anion translocation by these penetrat- were more complicated. In this case, the theoretical value of the ing cations [20]. diffusion potential could be observed only at acidic pH, the maxi- It was also found that SkQT1 stimulated State 4 respiration mal Dw being obtained at pH 4.0 (see Supplemental information, (Fig. S2A), which can be explained by discharge of Dw with electro- Fig. S1A and B). The effect was biphasic since Dw decayed with phoretic movement of SkQT1 and cycling of endogenous fatty acids time. If a pH gradient was increased across BLM, an H+ diffusion [15,20]. At high concentrations, SkQ1 and SkQT1 inhibited State 4 potential was generated (Fig. 2B). Such an effect was observed even and State 3 respiration of mitochondria utilizing both NAD+ sub- without adding fatty acids which were required when SkQ1 substi- strates and a-glycerophosphate (Fig. S2A–C). tuted for SkQTR1 [20]. These relationships can be explained by the In Fig. 4, effects of SkQT1 and SkQTR1 on cultures of human cells fact that rhodamine 19 (the cationic group of SkQTR1) per se can were tested. As shown in Fig. 4A, SkQTR1 specifically stains mito- operate as a protonophore, being a weak base (protonophorous chondria in intact human fibroblasts, resembling in this respect properties of SkQR1 were already described elsewhere [21]). Pro- previously studied SkQR1 [15–17].InFig. 4B, antiapoptotic effects tonophorous effect of SkQTR1 disappeared at acidic pH when the of SkQT1, SkQTR1, SkQ1, SkQR1, C12TPP and dodecyl rhodamine 19 concentration of deprotonated SkQTR1 became rate-limiting. This (C12R1) were compared. It is seen that the SkQTs are more efficient effect increased in time after creation of the transmembrane than the SkQs in preventing H2O2-induced apoptosis. They inhib- SkQTR1 gradient, most probably due to slow saturation by SkQTR1 ited apoptosis at lower concentrations and suppressed this process of the half-membrane leaflet facing the compartment of low more strongly than SkQs. Like other SkQs, SkQTs prevented frag- [SkQTR1]. mentation of elongated mitochondria (‘‘thread ? grain transi- In the next experiments, SkQT1 and related compounds were tion’’), accompanying apoptosis (not shown). studied in isolated mitochondria in vitro. SkQT1, like SkQ1 In the next series of experiments, we examined a novel effect of [15,16], was found to be a rechargeable antioxidant. Fig. 3A our antioxidants, i.e., inhibition of growth of human sarcoma cells. shows that the oxidized form of SkQT1 is reduced by the respira- One can see in Fig. 4C that low concentrations of SkQ1, SkQR1 and tory chain in an antimycin- and myxothiazol-sensitive manner. SkQT1 are inhibitory for a culture of human rhabdomyosarcoma. Similar properties are inherent in thymoquinone (Fig. 3B) and Effective concentrations were 0.02 nM, 0.2 nM and 2 nM for SkQ1 [15,16,22]. SkQ1, SkQR1 and SkQT1, respectively. Further study revealed that As is shown in Fig. 3C, SkQT1 prevents formation of malondial- non-targeted antioxidants can substitute for SkQ but at about 1 dehyde (MDA) in mitochondria treated with Fe2+ and ascorbate, million-fold higher concentrations (Trolox, 0.1 mM; N-acetyl cys- being almost 10- and 100-fold more active than SkQ1 and thymo- teine, 1 mM). The uncoupler FCCP (1 lM) completely abolished quinone, respectively. the SkQ inhibition. Dodecyltriphenylphosphonium (C12TPP) and Fig. 3D demonstrates the inhibiting effect of SkQT1 and SkQ1 on C12R1, penetrating cations lacking antioxidant (quinone) residue, H2O2 production by isolated rat heart mitochondria during the re- were inactive up to micromolar concentrations. Fibrosarcoma verse electron transfer from succinate to NAD+. It is seen that SkQ1 and osteosarcoma cells were also antioxidant-sensitive to some lowers this production, in line with our previous observations [17], degree, while human fibroblasts, cardiomyocites H9C2 and myo- depicting a pronounced optimum since an inhibition observed at blasts C2C12 appeared to be resistant. The mechanism of growth low SkQ1 concentrations changes to activation when the concen- inhibition consisted in (i) activation of apoptosis sensitive to tration increases. Experiments on SkQT1 also revealed such a bi- zVADfmk and (ii) arrest of the cell cycle at G2/M stage (not shown phasic action, the window between inhibiting and activating in figures). concentrations being much larger than that for SkQ1 (600 folds It should be stressed that the series of antioxidant active con- for SkQT1 vs 20 folds for SkQ1). centrations SkQs Trolox > N-acetyl cysteine is in line with our

Fig. 2. Responses of SkQT1 and SkQTR1 in BLM. To form BLM, synthetic diphythanoylphosphatidyl chlorine dissolved in decane was used. (A) Diffusion potential generated by 10-fold SkQT1 gradient. The response was initiated by adding 2 lM SkQT1 to one of compartments separated by BLM. Initial SkQT1 concentrations in both compartments was 0.2 lM. Incubation mixture, 1 mM KCl, 1 mM Tris-Mes, pH 6.5. (B) Protonophorous activity of SkQTR1. Incubation mixture, 300 nM SkQTR1, 1 mM KCl, 1 mM Tris, pH 8.5. Addition of KOH to one of the compartments shifted pH to 9.5. For details of the methods, see [15,64]. I.I. Severina et al. / FEBS Letters 587 (2013) 2018–2024 2021

Fig. 3. Effects of quinone derivatives on isolated mitochondria. (A) Reduction of SkQT1 by rat heart mitochondria. Mitochondria were isolated after Palmer et al. [65]. Incubation mixture, 2.5 mM succinate, 2 lM rotenone, 250 mM sucrose, 1 mM EDTA, 10 mM Mops-KOH, pH 7.4, mitochondria, 0.25 mg/ml. Reaction was initiated by adding 10 lM SkQT1 at zero time. Where indicated, 1 lM antimycin A or 1 lM myxothiazol were added. Aminco DW200 was employed to measure light absorbance at 270 nm. (B) Reduction of thymoquinone by mitochondria. Conditions as in (A) but thymoquinone was added instead of SkQT1. (C) Inhibition of malondialdehyde formation by rat heart mitochondria in the presence of Fe2+ and ascorbate. Amount of malondialdehyde was measured by means of its reaction with thiobarbituric acid (for procedure, see [15]). The light absorbance was read at 532 nm. (D) Effects of SkQ1 and SkQT1 on H2O2 production by rat heart mitochondria in State 4. Rat heart mitochondria (100 lg) were pre- incubated 1 min. at constant stirring at 25 °C in 2 ml solution containing 250 mM sucrose, 10 mM MOPS, pH 7.4, 1 mM EDTA, 5 lM Amplex Red. Then 5 units of horseradish peroxidase were added. Hydrogen peroxide production was initiated by subsequent addition of 10 mM succinate. Data presented as mean ± S.E. (E) Effect of SkQ1 and a mixture of SkQT1(p) and SkQT1(m) in proportion of 1.4:1 (SkQT1) on the membrane potential in rat heart mitochondria. Incubation mixture, 250 mM sucrose, 1 mM EDTA, 10 mM MOPS, 15 lM safranine O, 5 mM succinate and 2 lM rotenone. For other conditions and methods, see [15]. (F) SkQ1 and SkQT1 potentiate DW decrease caused by palmitate in yeast mitochondria. Incubation mixture, 0.6 M mannitol, 0.5 mM EDTA, 0.2 mM Tris–phosphate (pH 7.2), 20 mM succinate, 20 lM safranine O, and Yarrowia lipolytica mitochondria (0.5 mg protein 1 ml). For methods, see [5].

observations obtained in different experimental models [16,17,23]. antioxidant activity in isolated mitochondria and living cells are, However, this range inside the SkQ group, i.e., as a rule, higher than SkQ1. Historically, there is a principal differ- SkQ1 > SkQR1 > SkQTR1, is unusual (Fig. 4C). One of possible expla- ence between derivatives of plastoquinone and thymoquinone. The nations might consist in different rate of pumping of these com- formers were introduced as compounds of potential therapeutic pounds from the cell by multidrug resistance (MDR) ATPase [24]. activity several years ago whereas thymoquinone-containing black In fact, it was found (Fig. 4D and Fig. S3A and B) that SkQTR1 accu- cumin oil has been known as a medicine for thousand years, being mulation much smaller in the chemotherapy-resistant human mentioned in the Bible; it was described as the Melanthion of Hip- myeloid leukemia K562 cells. The accumulation was stimulated pocrates and Dioscorides and as the Gith of Pliny. Later, the Pro- by pluronic L61, an inhibitor of the MDR pump [25–27]. Compari- phet Muhammad advised: ‘‘Hold onto use the black cumin, for it son of accumulation of SkQR1 and SkQTR1 by cancer cells (K562, has a remedy for every illness except death’’ [29,30]. Fig. 4D, and Fig. S3A and B; Hela, Fig. 4E) and immortalized 3T3 Thymoquinone is a pre-dominant (25–60%) component of the fibroblasts (Fig. 4F) and by normal cells (human fibroblasts, essential oil Nigella sativa, a 20–30 cm tall herb belonging to Ran- Fig. 4G) clearly showed that the rate of this process is much slower unculaceae family [29,31,32]. It shows a very pleotropic favorable for SkQTR1 than for SkQR1 provided cancer cells with induced effect on various human diseases, being curative for osteosarcoma MDR pumps were studied. The difference between SkQTR1 and [33,34] and some other types of cancer [35–47], neuropathies SkQR1 strongly decreased in cancer cells without MDR pumps. [48,49], nephropathies [50,51], arthritis [51–53], inflammation Normal cells, such as fibroblasts, accumulate both compounds with [52–57], astma [58,59], sepsis [53], and decline in immune re- one and the same rate. As found by Gate et al. [28], methylation of sponses [29,35]. Remarkably, SkQs proved to be efficient in the anthracyclines inhibits the rate of their accumulation in cancer majority of these diseases [14,16–18,23,60,61]. The difference in cells due to the suppression of the activity of the MDR pump. This therapeutic effects thymoquinone and SkQs is quantitative. SkQs may explain the above-described relationships if we take into ac- operate at much lower concentrations and their action is usually count that SkQR1 can, in fact, be regarded as methylated derivative more pronounced than those of thymoquinone. All these relation- of SkQTR1 (see above, Fig. 1B). ships are hardly surprising. (i) Both SkQs [15,16] and thymoqui- none [29,35,62,63] are antioxidants. (ii) To some degree, 3. Comparison of effects of thymoquinone, SkQs and SkQTs thymoquinone, like SkQs, is specifically targeted to mitochondria since both thymoquinone and SkQs are effectively recharged by As shown in the preceding section, thymoquinone-like analogs reduction in center i of mitochondrial complex III [16,22]. This of SkQ1 (i) are mitochondria-targeted antioxidants and (ii) their means that in non-mitochondrial membranes oxidized (inactive) 2022 I.I. Severina et al. / FEBS Letters 587 (2013) 2018–2024

Fig. 4. Effects of SkQT1 and SkQTR1 on cell cultures. (A) SkQTR1 specifically stains mitochondria in human fibroblasts. Fibroblasts were incubated with 100 nM SkQTR1 for 45 min or 200 nM MitoTracker Green (mitochondria-specific fluorescent dye) for 15 min, and analyzed with an Axiovert microscope (Carl Zeiss) equipped with a Neofluar

100 NA 1.3 objective. (B) Effect of penetrating cations on H2O2-induced apoptosis. Human fibroblasts were pre-incubated for 24 h with various concentrations of SkQs and their analogs lacking quinone (C12TPP or C12R1). Then 0.5 mM H2O2 was added. The number of living cells was estimated 24 h after H2O2 addition. For methods, see [15]. (C) SkQs inhibit growth of the human rhabdomyosarcoma cells. Cells growth for one day or four days was measured in the absence (control) or in the presence of indicated concentration of SkQR1, SkQ1 or SkQT1. (D) K562 myeloid leukemia cells with or without induction of MDR pumps were studied. The cells were loaded with 50 nM SkQR1 or SkQTR1 for 90 min., washed and incubated for 30 min. Where indicated, pluronic L61 (plu) was added (60 lg/ml) on 10th min. before adding SkQ. Fluorescence was measured by FACS (Beckman-Coulter FC 500). (E) HeLa cells were studied. 100 nM SkQs and 10 lM FCCP were used. (F) 3T3 cells were studied. (G) Human fibroblasts were studied. In (E), (F) and (G), the samples were washed on 45th or 60th min. with a medium containing no SkQs. I.I. Severina et al. / FEBS Letters 587 (2013) 2018–2024 2023 forms of these quinones cannot be regenerated to their reduced [7] Smith, R.A., Porteous, C.M., Coulter, C.V. and Murphy, M.P. (1999) Selective (active) state. Therefore, in these membranes, quinol forms of targeting of an antioxidant to mitochondria. Eur. J. Biochem. 263, 709–716. [8] Kelso, G.F., Porteous, C.M., Coulter, C.V., Hughes, G., Porteous, W.K., SkQ or thymoquinone can act as antioxidant only once. (iii) Higher Ledgerwood, E.C., Smith, R.A. and Murphy, M.P. (2001) Selective targeting of efficiency of SkQs comparing to thymoquinone (see, e.g., Fig. 3C) is a -active ubiquinone to mitochondria within cells: antioxidant and explained by their electrophoretic accumulation of such cations as antiapoptotic properties. J. Biol. Chem. 276, 4588–4596. [9] Kelso, G.F., Porteous, C.M., Hughes, G., Ledgerwood, E.C., Gane, A.M., Smith, R.A. SkQs inside mitochondria [16,17] while thymoquinone cannot be and Murphy, M.P. (2002) Prevention of mitochondrial oxidative damage using accumulated being electroneutral. Moreover, SkQs are amphiphilic targeted antioxidants. Ann. N.Y. Acad. Sci. 959, 263–274. and, hence, have very high distribution coefficient between a [10] Saretzki, G., Murphy, M.P. and von Zglinicki, T. (2003) MitoQ counteracts telomere shortening and elongates lifespan of fibroblasts under mild oxidative membrane and a water phase [17]. This coefficient appears to be stress. Aging Cell 2, 141–143. much lower for the thymoquinone. Besides, the SkQs being pene- [11] Jauslin, M.L., Meier, T., Smith, R.A. and Murphy, M.P. (2003) Mitochondria- trating cations were shown to catalyze cycling, decreas- targeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous more effectively than untargeted antioxidants. FASEB J. 17, ing thereby Dw on mitochondrial membrane and inhibiting in this 1972–1974. way reactive oxygen species (ROS) production by respiratory chain [12] James, A.M., Cocheme, H.M., Smith, R.A. and Murphy, M.P. (2005) Interactions [20]. Thymoquinone lacking any cationic residues is incapable of of mitochondria-targeted and untargeted ubiquinones with the mitochondrial performing such a function. Thus, thymoquinone is less efficient respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools. J. Biol. Chem. antioxidant comparing with SkQT1 and SkQRT1 although it is, like 280, 21295–21312. SkQs, mitochondria-targeted antioxidant being rechargeable by [13] Murphy, M.P. and Smith, R.A. (2007) Targeting antioxidants to mitochondria the mitochondrial respiratory chain (Fig. 3B). It is not surprising, by conjugation to lipophilic cations. Annu. Rev. Pharmacol. Toxicol. 47, 629– 656. therefore, that the list of diseases cured by thymoquinone resem- [14] Skulachev, V.P. (2007) A biochemical approach to the problem of aging: bles that of the SkQ-cured diseases. It seems promising to con- ‘‘megaproject’’ on membrane-penetrating ions. The first results and prospects. struct a new generation of mitochondrial medicines on the basis Biochemistry (Moscow) 72, 1385–1396. [15] Antonenko, Y.N. et al. (2008) Mitochondria-targeted plastoquinone of SkQT1 which is more efficient than other SkQs. It is especially derivatives as tools to interrupt execution of the aging program. 1. Cationic interesting that SkQTR1 is a much better substrate for MDR pumps plastoquinone derivatives: synthesis and in vitro studies. Biochemistry of cancer cells. This pump expels SkQTR1 from cancer much faster (Moscow) 73, 1273–1287. [16] Skulachev, V.P. et al. (2009) An attempt to prevent senescence: a than SkQR1. In a normal cell, the amount of MDR pumps is negli- mitochondrial approach. Biochim. Biophys. Acta 1787, 437–461. gible and therefore SkQTR1 concentration in such cells should be [17] Skulachev, M.V. et al. (2011) Mitochondrial-targeted plastoquinone much higher than in tumors, an effect allowing to specifically pre- derivatives. Effect on senescence and acute age-related pathologies. Curr. Drug Targets 12 (6), 800–826. serve tissues other than tumors at anticancer therapies [24]. Inves- [18] Skulachev, V.P., Bogachev, A.V. and Kasparinsky, F.O. (2013) Principles of tigations into such a possibility are now in progress in our group. Bioenergetics, Springer, Berlin, Heidelberg. [19] Hutson, K.G. and Threlfall, D.R. (1980) Synthesis of plastoquinone-9 and phytylplastoquinone from homogentisate in lettuce . Biochim. Conflict of interest Biophys. Acta 632, 630–648. [20] Severin, F.F. et al. (2010) Penetrating cation/fatty acid anion pair as a M.V.S. is the general director of Mitotech LLC, a biotech com- mitochondria-targeted protonophore. Proc. Natl. Acad. Sci. USA 107, 663–668. [21] Antonenko, Y.N. et al. (2011) Derivatives of rhodamine 19 as mild pany which owns rights for compounds of SkQ type. V.P.S. is a mitochondria-targeted cationic uncouplers. J. Biol. Chem. 286, 17831–17840. board member of Mitotech LLC. [22] Skulachev, V.P. et al. (2010) Prevention of oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs). Biochim. Biophys. Acta 1797, 878–889. Ackowledgements [23] Neroev, V.V. et al. (2008) Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. This work was supported by the Research Institute of Mitoengi- SkQ1 returns vision to blind animals. Biochemistry (Moscow) 73, 1317–1328. neering of Lomonosov State University and by the Russian Founda- [24] Fetisova, E.K., Avetisyan, A.V., Izyumov, D.S., Korotetskaya, M.V., Chernyak, B.V. and Skulachev, V.P. (2010) Mitochondria-targeted antioxidant SkQR1 tion for Basic Research (Grant Nos. 12-04-31970, 13-04-01530, 12- selectively protects MDR (Pgp 170)-negative cells against oxidative stress. 04-00538). FEBS Lett. 584, 562–566. [25] Yusa, K. and Tsuruo, T. (1989) Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and Appendix A. Supplementary data transport of verapamil outward across the plasma membrane of K562/ADM cells. Cancer Res. 49, 5002–5006. Supplementary data associated with this article can be found, in [26] Kabanov, A.V., Batrakova, E.V. and Alakhov, V.Y. (2002) Pluronic block copolymers for overcoming drug resistance in cancer. Adv. Drug Delivery the online version, at http://dx.doi.org/10.1016/j.febslet.2013.04. Rev. 54, 759–779. 043. [27] Demina, T., Grozdova, I., Krylova, O., Zhirnov, A., Istratov, V., Frey, H., Kautz, H. and Melik-Nubarov, N. (2005) Relationship between the structure of Reference amphiphilic copolymers and their ability to disturb lipid bilayers. Biochemistry 44, 4042–4054. [28] Gate, L., Couvreur, P., Nguyen-Ba, G. and Tapiero, H. (2003) N-methylation of [1] Liberman, E.A. and Skulachev, V.P. (1970) Conversion of biomembrane- anthracyclines modulates their cytotoxicity and pharmacokinetics in wild produced energy into electric form IV. General discussion. Biochim. Biophys. type and multidrug resistant cells. Biomed. Pharm. 57, 301–308. Acta 216, 30–42. [29] Butt, M.S. and Sultan, M.T. (2010) Nigella sativa: reduces the risk of various [2] Liberman, E.A., Topaly, V.P., Tsofina, L.M., Jasaitis, A.A. and Skulachev, V.P. maladies. Crit. Rev. Food Sci. Nutr. 50, 654–665. (1969) Mechanism of coupling of oxidative phosphorylation and the [30] Malik Attaurrahman, S., He, C.H. and Clardy, J. (1985) Isolation and structure membrane potential of mitochondria. Nature 222, 1076–1078. determination of nigellicine, a novel alkaloid from the seeds of Nigella sativa. [3] Severin, S.E., Skulachev, V.P. and Iaguzhinskii, L.S. (1970) Possible role of Tetrahedron Lett. 26, 2759–2762. carnitine in the transport of fatty acids through the mitochondrial membrane. [31] Nickavar, A., Mojab, F., Javidnia, K. and Amolia, M.A.R. (2003) Chemical Biokhimiia 35, 1250–1253 (Russ.). composition of the fixed and volatile oils of Nigella sativa L. from Iran. Z. [4] Skulachev, V.P. (1988) Membrane Bioenergetics, Springer-Verlag, Berlin, New Naturforsch. 58, 629–631. York. [32] Mozaffari, F.S., Ghorbanli, M., Babai, A. and Sepehr, M.F. (2000) The effect of [5] Trendeleva, T.A., Sukhanova, E.I., Rogov, A.G., Zvyagilskaya, R.A., Seveina, I.I., water stress on the seed oil of Nigella sativa L. J. Essent. Oil Res. 12, 36–38. Ilyasova, T.M., Cherepanov, D.A. and Skulachev, V.P. (2012) Role of charge [33] Roepke, M., Diestel, A., Bajbouj, K., Walluscheck, D., Schonfeld, P., Roessner, A., screening and delocalization for lipophilic cation permeability of model and Schneider-Stock, R. and Gali-Muhtasib, H. (2007) Lack of p53 augments mitochondrial membranes. . http://dx.doi.org/10.1016/ thymoquinone-induced apoptosis and caspase activation in human j.mito.2012.10.006. osteosarcoma cells. Cancer Biol. Ther. 6, 160–169. [6] Burns, R.J., Smith, R.A. and Murphy, M.P. (1995) Synthesis and characterization [34] Ivankovic, S., Stojkovic, R., Jukic, M., Milos, M. and Jurin, M. (2006) The of thiobutyltriphenylphosphonium bromide, a novel thiol reagent targeted to antitumor activity of thymoquinone and thymohydroquinone in vitro and the mitochondrial matrix. Arch. Biochem. Biophys. 322, 60–68. in vivo. Exp. Oncol. 28, 220–224. 2024 I.I. Severina et al. / FEBS Letters 587 (2013) 2018–2024

[35] Woo, C.C., Kumar, A.P., Sethi, G. and Tan, K.H. (2012) Thymoquinone: potential [51] Budancamanak, M., Kanter, M., Demirel, A., Ocakci, A., Uysal, H. and Karakaya, cure for inflammatory disorders and cancer. Biochem. Pharmacol. 83, 443–451. C. (2006) Protective effects of thymoquinone and methotrexate on the renal [36] Gali-Muhtasib, H. et al. (2008) Thymoquinone reduces mouse colon tumor cell injury in collagen-induced arthritis. Arch. Toxicol. 80, 768–776. invasion and inhibits tumor growth in murine colon cancer models. J. Cell Mol. [52] Tekeoglu, I., Dogan, A. and Demiralp, L. (2006) Effects of thymoquinone Med. 12, 330–342. (volatile oil of black cumin) on rheumatoid arthritis in rat models. Phytother. [37] Kaseb, A.O., Chinnakannu, K., Chen, D., Sivanandam, A., Tejwani, S., Menon, M., Res. 20, 869–871. Dou, Q.P. and Reddy, G.P. (2007) Androgen receptor and E2F-1 targeted [53] Vaillancourt, F., Silva, P., Shi, Q., Fahmi, H., Fernandes, J.C. and Benderdour, thymoquinone therapy for hormone-refractory prostate cancer. Cancer Res. M. (2011) Elucidation of molecular mechanisms underlying the protective 67, 7782–7788. effects of thymoquinone against rheumatoid arthritis. J. Cell Biochem. 112, [38] Banerjee, S., Kaseb, A.O., Wang, Z.W., Kong, D.J., Mohammad, M., Padhye, S., 107–117. Sarkar, F.H. and Mohammad, R.M. (2009) Antitumor activity of gemcitabine [54] Chehl, N., Chipitsyna, G., Gong, Q., Yeo, C.J. and Arafat, H.A. (2009) Anti- and oxaliplatin is augmented by thymoquinone in pancreatic cancer. Cancer inflammatory effects of the Nigella sativa seed extract, thymoquinone, in Res. 69, 5575–5583. pancreatic cancer cells. HPB (Oxford) 11, 373–381. [39] Jafri, S.H., Glass, J., Shi, R.H., Zhang, S.L., Prince, M. and Kleiner-Hancock, H. [55] Sethi, G., Ahn, K.S. and Aggarwal, B.B. (2008) Targeting nuclear factor-kappa B (2010) Thymoquinone and cisplatin as a therapeutic combination in lung activation pathway by thymoquinone: role in suppression of antiapoptotic cancer: in vitro and in vivo. J. Exp. Clin. Cancer Res. 29. gene products and enhancement of apoptosis. Mol. Cancer Res. 6, 1059–1070. [40] Badary, O.A., Al-Shabanah, O.A., Nagi, M.N., Al-Rikabi, A.C. and Elmazar, M.M.A. [56] El Gazzar, M., El Mezayen, R., Nicolls, M.R., Marecki, J.C. and Dreskin, S.C. (1999) Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in (2006) Downregulation of leukotriene biosynthesis by thymoquinone mice by thymoquinone. Eur. J. Cancer Prev. 8, 435–440. attenuates airway inflammation in a mouse model of allergic asthma. [41] Badary, O.A., Nagi, M.N., Al-Shabanah, O.A., Al-Sawaf, H.A., Al-Sohaibani, M.O. Biochim. Biophys. Acta 1760, 1088–1095. and Al-Bekairi, A.M. (1997) Thymoquinone ameliorates the nephrotoxicity [57] Marsik, P., Kokoska, L., Landa, P., Nepovim, A., Soudek, P. and Vanek, T. (2005) induced by cisplatin in rodents and potentiates its antitumor activity. Can. J. In vitro inhibitory effects of thymol and quinones of Nigella sativa seeds on Physiol. Pharmacol. 75, 1356–1361. cyclooxygenase-1- and -2-catalyzed prostaglandin E2 biosyntheses. Planta [42] Badary, O.A. (1999) Thymoquinone attenuates ifosfamide-induced Fanconi Med. 71, 739–742. syndrome in rats and enhances its antitumor activity in mice. J. [58] El-Mahmoudy, A., Shimizu, Y., Shiina, T., Matsuyama, H., El-Sayed, M. and Ethnopharmacol. 67, 135–142. Takewaki, T. (2005) Successful abrogation by thymoquinone against induction [43] Norwood, A.A., Tan, M., May, M., Tucci, M. and Benghuzzi, H. (2006) of diabetes mellitus with streptozotocin via nitric oxide inhibitory Comparison of potential chemotherapeutic agents, 5-fluoruracil, green tea, mechanism. Int. Immunopharmacol. 5, 195–207. and thymoquinone on colon cancer cells. Biomed. Sci. Instrum. 42, 350–356. [59] Mansour, M. and Tornhamre, S. (2004) Inhibition of 5-lipoxygenase and [44] Salomi, N.J., Nair, S.C., Jayawardhanan, K.K., Varghese, C.D. and Panikkar, K.R. leukotriene C4 synthase in human blood cells by thymoquinone. J. (1992) Antitumour principles from Nigella sativa seeds. Cancer Lett. 63, 41–46. Inhib. Med. Chem. 19, 431–436. [45] Yi, T. et al. (2008) Thymoquinone inhibits tumor angiogenesis and tumor [60] Bakeeva, L.E. et al. (2008) Mitochondria-targeted plastoquinone derivatives as growth through suppressing AKT and extracellular signal-regulated kinase tools to interrupt execution of the aging program. 2. Treatment of some ROS- signaling pathways. Mol. Cancer Ther. 7, 1789–1796. and age-related diseases (heart arrhythmia, heart infarctions, kidney [46] Harzallah, H.J., Grayaa, R., Kharoubi, W., Maaloul, A., Hammami, M. and ischemia, and stroke). Biochemistry (Moscow) 73, 1288–1299. Mahjoub, T. (2012) Thymoquinone, the Nigella sativa bioactive compound, [61] Agapova, L.S. et al. (2008) Mitochondria-targeted plastoquinone derivatives as prevents circulatory oxidative stress caused by 1,2-dimethylhydrazine in tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 erythrocyte during colon postinitiation carcinogenesis. Oxid. Med. Cell on tumor development from p53-deficient cells. Biochemistry (Moscow) 73, Longev. 2012, 854065. 1300–1316. [47] Attoub, S. et al. (2012) Thymoquinone as an anticancer agent: evidence from [62] Mansour, M.A., Nagi, M.N., El-Khatib, A.S. and Al-Bekairi, A.M. (2002) Effects of inhibition of cancer cells viability and invasion in vitro and tumor growth thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT- in vivo. Fundam. Clin. Pharmacol.. http://dx.doi.org/10.1111/j.1472- diaphorase in different tissues of mice: a possible mechanism of action. Cell 8206.2012.01056.x. Biochem. Funct. 20, 143–151. [48] Kanter, M. (2008) Effects of Nigella sativa and its major constituent, [63] Badary, O.A., Taha, R.A., El-Din, A.M.G. and Abdel-Wahab, M.H. (2003) thymoquinone on sciatic nerves in experimental diabetic neuropathy. Thymoquinone is a potent anion scavenger. Drug Chem. Toxicol. Neurochem. Res. 33, 87–96. 26, 87–98. [49] Mohamed, A., Shoker, A., Bendjelloul, F., Mare, A., Alzrigh, M., Benghuzzi, H. [64] Severina, I.I. (1982) Nystatin-induced increase in photocurrent in the system and Desin, T. (2003) Improvement of experimental allergic encephalomyelitis bacteriorhodopsin proteoliposome bilayer planar membrane. Biochim. (EAE) by thymoquinone; an oxidative stress inhibitor. Biomed. Sci. Instrum. Biophys. Acta 681, 311–317. 39, 440–445. [65] Palmer, J.W., Tandler, B. and Hoppel, C.L. (1977) Biochemical properties of [50] Kanter, M. (2009) Protective effects of thymoquinone on streptozotocin- subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac induced diabetic nephropathy. J. Mol. Histol. 40, 107–115. muscle. J. Biol. Chem. 252, 8731–8739.